The present invention relates to isolated polynucleotide molecules useful for analyzing alloantigen phenotypes, to peptides encoded by these molecules, and to the diagnostic and therapeutic uses thereof relating to the "Pen" human platelet polymorphism. Among such uses is a method for typing platelet membrane glycoproteins which entails an analysis either of genomic DNA or of amplified cDNA produced from platelet mRNA.
Blood obtained from different individuals has been found to have different antigenic and immune properties, to the extent that antibodies in the blood of one person may react with antigens on red blood cells or platelets in the blood of another individual. These antigens are often found on membrane glycoproteins present on the surface of the cells. These membrane glycoprotein antigens can induce the production of antibodies against them when they are introduced as foreign proteins in transfused blood or in fetal blood. Human platelets and red blood cells contain dozens of identifiable membrane glycoprotein constituents, only some of which have been well characterized.
Membrane glycoproteins which induce antibody production in the same species are called "alloantigens." Alloantigens have been characterized for both red blood cells and platelets. Recognized classes of red blood cell and platelet alloantigens have been described, over the past 30 years, based on observations of antibody reactions occurring when patients have been exposed to blood from other individuals.
One system of alloantigens, consisting of the platelet Pen.sup.a and Pen.sup.b alloantigens, are carried by the human platelet membrane glycoprotein IIb-IIIa (GPIIb-IIIa) complex, which mediates platelet aggregation by providing functional receptors for fibrinogen on platelet surfaces. See Friedman & Aster, Blood 65:1412-1415 (1985). Further investigation has revealed that the Pen alloantigen system is located on GPIIIa. See Shibata & Mori, Proc. Japan Acad. 63:36-38 (1987).
GPIIIa is known to bear at least one other clinically important, alloantigenic determinant, Pl.sup.A, which is responsible for eliciting an immune response in two well-described clinical syndromes, post-transfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NATP). See Kunicki & Newman in CURRENT STUDIES IN HEMATOLOGY AND BLOOD TRANSFUSION 18-32 (1986); Aster in ADVANCES IN IMMUNOLOGY AND BONE MARROW TRANSPLANTATION 103-118 (1984). While polymorphisms such as Pl.sup.A on GPIIIa and Bak located on GPIIb are most often implicated in PTP and NATP in Caucasian and black populations, the Pen alloantigen system is the most frequent cause of these disorders in oriental individuals. See Shibata & Mori, Proc. Japan Acad. 63:36-38 (1987); Furihata et al., J. Clin. Invest. 80:1624-1630 (1987).
There are two serologically defined allelic forms of the Pen alloantigen which are designated "Pen.sup.a " and "Pen.sup.b." The location of the Pen antigen system, like that of Pl.sup.A, has been shown to be associated with the GPIIIa molecule. See Shibata & Mori, loc. cite and Furihata et al. loc. cite. The gene frequencies for these two alleles in the Japanese population have been calculated to be 99.8% for Pen.sup.a and 0.2% for Pen.sup.b. See Shibata et al., Vox Sang. 51:334 (1986); Simon et al., Amer. J. Hematol. 29:38-40 (1988).
The immunological characteristics of blood group alloantigens have often been attributed to the extensive glycosylation of these proteins. Differences in glycosylation may be due to either generic variation among glycosylation enzymes, as in the ABO alloantigen system, or to amino acid-sequence polymorphisms among the alloantigens themselves, or to a combination of these factors as in the MN system. See Eisen et al., IMMUNOLOGY (2d ed. 1980). In particular, differences in specific sialic acid structures have been determined to contribute to the expression of actual allogenic epitopes. See Sadler et al., J. Biol. Chem. 254(6):2112-2119 (1979); Take et al., British J. Haematol. 76:395-400 (1990). The basis for the variations responsible for the relevant epitopes has not yet been reported for either the Pena or Pen.sup.b forms of the 100 kilodalton (kd) GPIIIa molecule.